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BIOC3008 > Biofuels: The Future for Energy Production? > Flashcards

Biofuels: The Future for Energy Production? Flashcards

1
Q

Why is it crucial we obtain a sustainable way of attaining fuel?

A

It is predicted oil will run out in the next 50-100 years (Stephenson et al. 2010).
The oil that remains is predominantly in the Middle East (66%) , thus the West is concerned about their ‘Energy Secutiy’
Oil is a finite resource, thus must attain a renewable source of energy, specifically energy-dense fuels.

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2
Q

What are the first generation of biofuels?

A

Biodiseal and bioethanol.

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3
Q

What is biodiseal and bioethanol?

A

Biodiseal is derived from storage lipids such as triacylglycerides (TAGs), which can be extracted from oil rich crops such as soybean, rapeseed, jatropha and pal oil. The TAGs are then transesterified to makes FAMEs.
Bioethanol is obtained from sugarcane and cor, sugar and starch are extracted, they undergo hydrolysis and then fermentation to yield ethanol.

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4
Q

What are TAGs?

A

Triacylglycerides - which are transesterified to form fatty acid methyl esters (FAMEs) and glycerol

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5
Q

What are FAMEs

A

Fatty acid methyl esters - which can be separated from glycerol to form biodiseal

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6
Q

What are second generation biofuels?

A

Via enzyme hydrolyssis of lignocellulose of cell walls, to yield pentose sugar which can then be fermented to form ethanol. These are obtained from Misccanthus or willow.

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7
Q

What are challenges faced by 2nd generation of biofuels?

A

The breakdown of lignocellulose to fermentable products, particularly as plant cells have evolved to be resistant to enzymatic degradation.

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8
Q

What are the negatives of 2nd generation biofuels?

A

Require the use of vast amounts of land. Highly energy expensive to chop down trees and transport crops.

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9
Q

What are third generation of biofuels?

A

Biogases

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10
Q

What are biogases?

A

These can be obtained from anaerobic digestion of organic materials, such as waste paper and soft plant tissue, food waste, sewage and animal waste.
These can produce Methane (50-75%), CO2 (20-45%) and Hydrogen (5%).

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11
Q

How are biogases created?

A

Insertion of waste into a anaerobic bioreactor, which digests the waste materials in four main stages:

  1. Hydrolysis to produce FAs, AAs and Sugars
  2. Acidogenesis to generate Carbonic acids and alcohols, Ammonia, H2 and CO2
  3. Acetogenesis to produce Acetic acid, CO2 and H2
  4. Methanogenesis –> CH2 and CO2
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12
Q

What are the major issue of growing crops for biofuels?

A
  1. Requires huge amounts of fertile land - Soyabeans produce 560 litre/hectare
  2. Most land is currently used for agriculture, issue with growing population
  3. Life Cycle Analysis (LCA) suggests energy input is greater than output (transport/creation of fertile land)
  4. Cultivation required irrigation with fresh water and fertilisers
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13
Q

What is algae?

A

Wide range of simple aquatic organisms that typically carry out oxygenic photosynthesis, can either be macroscopic or microscopic and typically grow in a wide range of environments, particularly environments which would otherwise not be cultivable e.g. hypersaline lakes

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14
Q

What are the advantages of algae in the production of biofuels?

A
  1. Rapid Growth Rates - double biomass in hours/days instead of weeks
  2. Some species hold extremely high oil content (>50% of dry mass), although hard to extract
  3. Manipulate to increase biomass and oil production (dilution of N-free culture)
  4. Can be grown without the exploitation of ‘useful’ land
  5. Don’t require expensive irrigation
  6. Can be linked to CO2 capture
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15
Q

How can algae be cultivated?

A
  1. Algal farms/Open ponds
  2. Closed photobioreactor (PBRs)
  3. Off-shore cultivation
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16
Q

What are PBRs?

A

Photobioreactors; facilitate increased control of growth conditions, e.g. temp, light, CO2 content. However are extremely expensive, harder to scale, problems with O2 build up.

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17
Q

What are the adv and neg of Open Ponds?

A

+ive - cheap, easy to scale
-ive - problems with eco-contamination (GMs), problems with controlling conditions, problems with contamination/competition/predation

18
Q

What is the disadvantage of using algae to generate biofuel?

A

Biomass ultimately limited by shading problems and photo-inhibition. Thus requires a means of mechanically mixing the system, decreasing overall energy output in LCA, hard to harvest and de-water

19
Q

What methods can be implemented in algal production to improve their production?

A
  1. Flocculation of algal cells - means of aggregation to separate algae from water (de-watering system, often too expensive)
  2. Capillary based system - reduce the issue of photo-inhibition
  3. Algae Venture Systems Harvester - utilise gravity as the water feed
  4. Mutants PSII affecting the antenna - allows for further penetration of light
  5. Decrease biosynthesis of starch to increase biomass production and thus TAG production
20
Q

How can light capture be improved?

A

NB - 95% of light is lost from sunlight.

  1. Rapid mixing of culture at 1Hz (energy inefficient)
  2. Use of mutants with reduced light-harvesting abilities
21
Q

What mutations can alter light-harvesting abilities?

A

Those which effect the antenna of the PSII to reduce photoinhibition and allow greater light penetration. Mutants generated following a variant screen. TAM2 - improved by 20% improvement in biomass production.

22
Q

What is photoinhibition?

A

Photoinhibition is light-induced reduction in the photosynthetic capacity of a plant, alga, or cyanobacterium. Photosystem II (PSII) is more sensitive to light than the rest of the photosynthetic machinery, and most researchers define the term as light-induced damage to PSII.

23
Q

How can we improve TAG production?

A
  1. Starch Biosynthesis

2. Overexpression of key TAG biosynthesis genes during normal growth

24
Q

How does inhibition of starch biosynthesis result in improved TAG synthesis?

A

Low biomass and high TAG production are both coupled with high starch biosynthesis, induced under nutrient stress. Low starch biosynthesis is additionally coupled with high biomass levels under normal growth conditions. Thus inhibition of starch biosynthesis under nutrient stress allows for a higher biomass production.
Use a simple iodine screen to recognise starch mutants (don’t stain dark blue).

25
Q

Which genes should be overexpressed to result in increased TAG production?

A

Diacylglycerol acyl transferase (DAGT) - last enzyme in biosynthetic pathway of TAG
AcetylCoA carboxylase - the rate limiting step
Attempts underway to recognise the ‘master’ switch that senses nutrient stress and triggers the upregulation of TAG biosynthetic pathway

26
Q

What is the issue with TAG production from algae?

A
  1. Biomass must be harvested and dewatered prior to extraction
  2. Cells must be broken and organic solvents used to extract TAGs (energy inefficient)
  3. TAGs must be converted to FAMEs (no direct attainment of biofuels)
27
Q

What is the ‘ideal algae’

A

Chlamydomonas reinhardtii

28
Q

Why is C.reinhardtii thought of as the ideal organism?

A
  1. Fast growing species requiring a basic growth medium
  2. Generally Recognised as Safe (GRAS)
  3. Capable of both phototrophic and heterotrophic growth
  4. Genome entirely sequenced
  5. Has simple sex cycle
  6. Strains can be frozen to prevent genetic drift
29
Q

What can we used as a foreign expression unit?

A

Chloroplast

30
Q

Why is a chloroplast used as a foreign expression unit?

A
  1. Natural storage compartment allowing for the hyper-accumulation of foreign protein
  2. Generation of chloro. transformant is quick
  3. Chloroplast genome can be targetted
  4. Could express multiple foreign genes as an operon
  5. Strong endogenous promoters
  6. No gene silencing mechanisms present in the chloroplast
31
Q

How can genes be delivered to the chloroplast?

A

Shotgun methods - coat gold particles with DNA, shoot at a law of 10(^7) Chlamydomonas, giving rise to 10-100 transformants

32
Q

What are a new line of antibacterials?

A

Bacteriophages

33
Q

How are mutants with reduced light-harvesting abilities screened?

A
  1. Early log phase culture are subject to 6W/245nm of UV radiation (10% survival) - 20 mins
  2. Left in the dark for 2 hours, survivors plated on acetate
  3. Undergo fluorescence analysis and chlorophyll a:b analysis
  4. Ideal mutants selected following Illumina sequences and mutant analysis
34
Q

How are starch mutants selected for?

A
  1. UV irradiation – to ensure photo-inhibition mutants
  2. These were plated onto low N medium
  3. Colonies were stained with iodine vapor
  4. Re-streak colonies that don’t stain dark blue, thus don’t contain starch as simple carbohydrates oxidize the Iodine to a dark blue color
  5. Repeat iodine staining to confirm phenotype
  6. Mutants found to accumulate more TAGs
35
Q

What TAG synthesis genes are overexpressed?

A
  1. Diacylglycerol acyl transferase (DAGAT) - the final enzyme in TAG boisynthetic pathway.
  2. AcetylCoA Catalase - which is a rate-limiting enzyme (due to low conc.) in the pathway.
  3. Attempts have been made to identify the ‘master switch’ that sense N limitation and triggers the up-regulation of the whole TAG biosynthetic pathway
36
Q

What are the issues with using TAGs as biofuels?

A
  1. TAGs require further processing - transesterification to produce FAMEs and a separation protocol to separate glycerol and FAMEs
  2. Using algae to make TAGs requires energy expensive processes, such as dewatering
  3. Cells must be broken and organic solvents used to extract, again further processing requiring more agents, depletes the total energy obtained from the protocol
37
Q

Describe the integration gene of a chloroplast vector.

A

Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination, which is aided by a RecA type.

38
Q

Describe the insertion site in the chloroplaset genome.

A

Transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5′ or 3′ untranslated regions (UTRs) or promoters. To date, the most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the chloroplast genome.

39
Q

What is the composition of wood?

A

Cellulose - Unbranched glucose polymer
Hemicellulose - branch polymers composed of glucose plus various hexose and pentose sugars
Lignin- complex and heterogenous biopolymer

40
Q

What are other alternative fuels we could obtain?

A

Fatty alcohols - (C16-18), introduction of plant enzyme FAR might allow precursor fatty acylCoA to be transformed to fatty alcohol.
Isoprenoids - are a large family of branched unsaturated carbons. The introduction of isoprene synthase gene (IS) showed isoprene to accumulate in Chlamydomonas.
Alkanes - plants make long chain alkanes, some algae and cyanobacteria make using fatty acid reductase (FAR).

41
Q

What gene is deleted during chloroplast transformation?

A

psbH - small subunit of PSII

42
Q

How are the transformants selected?

A

delta psbH cells, the expression vector will contain psbH and will recover them to photoautotrophic growth.

BIOC3008 (4 decks)

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